Immunohistochemistry Anti-Glial Fibrillary Acidic Protein (GFAP) Mouse Human

Get tips on using Human FABP2 / Fatty Acid-Binding Protein, Intestinal ELISA Kit to perform ELISA Human - FABP2

Get tips on using Human/Mouse/Rat Phospho-Akt (S473) Pan Specific Antibody to perform Western blotting AKT

Get tips on using Quant-iT™ RNA Assay Kit to perform RNA quantification Fuorimetric - mouse glial cells

Protein expression refers to the techniques in which a protein of interest is synthesized, modified or regulated in cells. The blueprints for proteins are stored in DNA which is then transcribed to produce messenger RNA (mRNA). mRNA is then translated into protein. In prokaryotes, this process of mRNA translation occurs simultaneously with mRNA transcription. In eukaryotes, these two processes occur at separate times and in separate cellular regions (transcription in nucleus and translation in the cytoplasm). Recombinant protein expression utilizes cellular machinery to generate proteins, instead of chemical synthesis of proteins as it is very complex. Proteins produced from such DNA templates are called recombinant proteins and DNA templates are simple to construct. Recombinant protein expression involves transfecting cells with a DNA vector that contains the template. The cultured cells can then transcribe and translate the desired protein. The cells can be lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield. These expression systems include mammalian, insect, yeast, bacterial, algal and cell-free. Each of these has pros and cons. Mammalian expression systems can be used for transient or stable expression, with ultra high-yield protein expression. However, high yields are only possible in suspension cultures and more demanding culture conditions. Insect cultures are the same as mammalian, except that they can be used as both static and suspension cultures. These cultures also have demanding culture conditions and may also be time-consuming. Yeast cultures can produce eukaryotic proteins and are scalable, with minimum culture requirements. Yeast cultures may require growth culture optimization. Bacterial cultures are simple, scalable and low cost, but these may require protein-specific optimization and are not suitable for all mammalian proteins. Algal cultures are optimized for robust selection and expression, but these are less developed than other host platforms. Cell-free systems are open, free of any unnatural compounds, fast and simple. This system is, however, not optimal for scaling up.

Get tips on using Human Retinol binding protein 4 ELISA Kit (RBP4) (ab108897) to perform ELISA Human - RBP4

Get tips on using Human Breast Cancer Susceptibility Protein 2 (BRCA2) ELISA Kit to perform ELISA Human - BRCA2

Get tips on using Anti-phospho-Histone H2A.X (Ser139) Antibody to perform TissueFAxs phospho-Histone H2A.X (Ser139) - Mouse Human

Get tips on using Anti-phospho-Histone H2A.X (Ser139) Antibody to perform Immunofluorscence  phospho-Histone H2A.X (Ser139) - Mouse Human

Get tips on using Human/Mouse/Rat Activin A Quantikine ELISA Kit to perform ELISA Rat - Activin

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.